Development of an In Vitro Diagnostic Technique for Malassezia furfur A thesis submitted in partial fulfilment of the requirement for the Degree of Master of Science in Microbiology in the University of Canterbury by Stephanie Fiona ~uick University of Canterbury vw+ of Contents 1 ! :) TABLE OF CONTENTS Ta hie of Contents ............................................................ 8 •••••••• 0 ••• "." ••••••••••• 1 List of Tables ............................. o ••••••• o ••••••••••••••••••••••••••••• 0 •••••••••• 0 •••••••••••••• 7 List of Figures ........................................................................................... 8 Abstract............................. o ••••• 0 ••• 0' ••••••••••••••••••••••••••••••••••••••••••••••••••••• 00 •••••• 9 Acknowledgemen ts ..... o ••••• e •••••••••••• e •••••••••••••••••••••••••••••••••••••••• o ••••• o ••••••• e •• l 0 1. INTRODUCTION .o, •• o •••••••• e •••• O' •••• O •••••••••••••••••••••••••••••••••••••••••••••••••••••• 11 1.1 THE GENUS MALASSEZIA ..................................................................... 11 1.2 TAXONOMY OF MALASSEZIA FURFUR .................................................. 12 1.3 MORPHOLOGy .................................................................................... 15 1.4 PATHOGENICITY ................................................................................. 18 1.5 fIABITAT ............................................................................................ 20 1.6 GROWTH OF MALASSEZIA FURFUR IN VITRO ......................................... 22 1.7 IDENTIFICATION ................................................................................. 24 1.8 CONTROL ........................................................................................... 25 1.9 ANTIMICROBIAL COMPOUNDS PRODUCED BY MALASSEZIA FURFUR ... 26 2. MATERIALS AND METHODS ..................................... e ••••••••••••••••• 27 2.1 CULTURE ............................................................................................ 27 Table of Contents 2 2.1.1 Culturing o/Freeze-Dried Isolate ............................................................... 27 2.1.2 Culturing o/Skin Isolate ............................................................................. 28 2.2 IDENTIFICATION METHODS FOR MALASSEZIA FURFUR ......................... 29 2.3 GROWTH MEDIA ................................................................................ 30 2.3.1 Media Incorporating Indicators .................................................................. 31 2.3.1.1 Utilisation of Urea ............. 88 ••• "& •••••• 08 •••••• 08 •••• '11 ••••••••••••••••••••••• 111 •••••••••••••••••• 31 2.3.1.2 Utilisation of Nitrogen Sources ............................................................ 31 2.3.1.3 Utilisation of Amides ............................................................................ 032 2.3.2 Media Incorporating Lipids ........................................................................ 32 2.3.2.1 Utilisation of Some PoJyoxyethylene Sorbitan Esters (Tween ~ ...... 32 2.3.2.2 Utilisation of Cholesterol, Egg Yolk and Milk .................................. 33 2.3.3 Media Incorporating Other Nutrients ......................................................... 33 2.3.3.1 Growth on Sweat Medium.................................................................... 33 2.3.3.2 Utilisation of Taurocholic Acid ............................................................ 34 2.3.3.3 Assessment of Substrate Assimilation System ....................... 34 2.3.31104 Gelatine Hydrolysis .... o •••••••••••• ooov •• "' .............................. iI .. " ......................... O"" ••• 34 2.4 INDUCTION OF THE HYPHAL PHASE OF MALASSEZIA FURFUR .............. 35 2.5 RESPONSE TO INHIBITORY AGENTS .................................................... 36 2.5.1 Antifungal Agents ........................................................................................ 36 2.5.1.1 Amphotericin B Sensitivity .. 0 •••• 0 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 36 2.5.1.2 Nystatin Sensitivity ................ 0 ••••••••••••••••••• 0 •••••••••••••••••••••••••••••••••••••••••• 37 2.5.1.3 Thiarubrine A Sensitivity ........................................................................... 37 2.5.1.4 Agricultural Fungicide Sensitivity ....................................................... 38 2.5.2 Effect o/Fungal Cell Wall Degrading Enzymes ......................................... 39 Table of Contents 3 2.6 ANTIMICROBIAL AGENTS PRODUCED BY MALASSEZIA FURFUR ........... 41 2.6.1 Azelaic Acid Assay ...................................................................................... 41 2.6.2 Ethyl Acetate Extraction ofFungal Metabolites from Malasseziafurfur ... 43 3. RESULTS ................................................... 0 •••••••••••••••••••••••••••••••••••••••• 45 3.1 CULTURE ............................................................................................ 45 3.1.1 Culturing ofFreeze-Dried Isolate ............................................................... 45 3.1.2 Culturing ofSkin Isolate ............................................................................. 45 3.2 IDENTIFICATION METHODS FOR MALASSEZIA FURFUR ......................... 45 3.3 GROWTH MEDIA ................................................................................ 46 3.3.1 Media Incorporating Indicators .................................................................. 46 3.3.1.1 Utilisation of Urea ................................................................................. 46 3.3.1.2 Utilisation of Nitrogen Sources ............................................................ 49 3.3.1.3 Utilisation of Amides ............................................................................. 49 3.3.2 Media Incorporating Lipids ........................................................................ 52 3.3.2.1 Utilisation of Polyoxyethylene Sorbitan Esters (Tween®) .................. 52 3.3.2.2 Utilisation of Cholesterol, Milk and Egg Yolk ................................... 53 3.3.3 Media Incorporating Other Nutrients ......................................................... 54 3.3.3.1 Growth on Sweat Medium .................................................................... 54 3.3.3.2 Utilisation of Taurocholic Acid ............................................................ 54 3.3.3.3 Assessment of ID 32C Substrate Assimilation System ....................... 57 3.3.3.4 Gelatine Hydrolysis ............................................................................... 58 3.4 INDUCTION OF THE HYPHAL PHASE OF MALASSEZIA FURFUR .............. 59 3.5 REACTION TO INHIBITORY AGENTS .................................................... 60 Table of Contents 4 3.5.1 Antifungal Agents ........................................................................................ 60 3.5.1.1 Amphotericin B Sensitivity ."" ............................................................... 60 3.5.1.2 Nystatin Sensitivity .............. ,.................................................................. 61 3.5.1.3 Thiarubrine A Sensitivity .............................................. "....... 0 •••••• " ••••••• 64 3.5.1.4 Agricultural Fungicide Sensitivity ....................... u .............................. 67 3.5.2 Effect ofFungal Cell Wall Degrading Enzymes ......................................... 69 3.6 ANTIMICROBIAL AGENTS PRODUCED BY MALASSEZIA FURFUR ............ 72 3.6.1 Azelaic Acid Assay ...................................................................................... 72 3.6.2 Ethyl Acetate Extraction ofFungal Metabolites ofMalasseziafurfur ....... 72 4. DISCUSSION ............••..••...••.•.•..•.....•..••.••••..•....•......•......•... ,,0 •••••••••••••• 73 4.1 CULTlTRE AND GROWTH ..................................................................... 73 4.2 NUTRIENT REQUIREMENTS ................................................................. 74 4.3 IDENTIFICATION AND CONTROL ......................................................... 76 4.4 FlTTUREREsEARCH ............................................................................ 79 4.5 SUMMARy ...................................................................................... 80 5. REFEREN CES ...........•....•...............•..•........•...•.................... 1) ••••••••••••• 81 6. APPENDIX OF MEDIA ............... D •••••••••••••••••••••••••••••••••••••••••••••••••••• 92 6.1 DIXON'S AGAR ................................................................................... 92 6.2 DIXON'S BROlli ................................................................................. 92 6.3 DIXON'S AGAR WIlli CYCLOHEXIMIDE, CHLORAMPHENICOL AND GENTAMICIN (CCG) ................................................................................. 93 Table of Contents 5 6.4 SABOURAUD DEXTROSE AGAR (SDA) ............................................... 94 6.5 SDA WITH OLIVE OIL ......................................................................... 94 6.6 SDA WITH TWEEN SO® AND GLyCEROL ............................................. 95 6.7 SDA WITH TWEEN SO®, GLYCEROL AND CCG .................................. 95 6.S CHRISTENSEN'S UREA AGAR ............................................................. 96 6.9 CHRISTENSEN'S UREA AGAR WITH TWEEN SO® AND GLYCEROL ....... 97 6.10 UREA AGAR BASE .......................................................................... 97 6.11 PHENOL RED BROTH BASE ............................................................